Immunogenic compositions for protection against Chlamydial infection

ABSTRACT

A protective immune response against Chlamydial infection is achieved by in vivo administration of an immunogenic composition comprising two vectors and a pharmaceutically-acceptable carrier therefor. One of the vectors comprises a first nucleotide sequence encoding a major outer membrane protein (MOMP) of a strain of  Chlamydia,  preferably  C. pneumoniae,  and a promoter sequence operatively coupled to the first nucleotide sequence for expression of the MOMP in the host. The other of the vectors comprises a second nucleotide sequence encoding a 76 kDa protein of a strain of  Chlamydia,  preferably  C. pneumoniae,  and a promoter sequence operatively coupled to the second nucleotide sequence for expression of the 76 kDa protein in the host. The protection efficiency which is achieved by the immunization procedure is enhanced over that attained with the individual vectors alone.

FIELD OF THE INVENTION

The present invention relates to immunogenic compositions for protectionagainst disease caused by Chlamydia infection in mammals, includinghumans.

BACKGROUND OF THE INVENTION

Chlamydiae are procaryotes. They exhibit morphologic and structuralsimilarities to gram negative bacteria, including a trilaminar outermembrane, which contains lipopolysaccharide and several membraneproteins Chlamydiae are differentiated from other bacteria by theirmorphology and by a unique developmental cycle. They are obligateintracellular parasites with a unique biphasic life cycle consisting ofa metabolically inactive but infectious extracellular stage and areplicating but non-infectious intracellular stage. The replicativestage of the life-cycle takes place within a membrane-bound inclusionwhich sequesters the bacteria away from the cytoplasm of the infectedhost cell.

Because chlamydiae are small and multiply only within susceptible cells,they were long thought to be viruses. However, they have manycharacteristics in common with other bacteria: (1) they contain both DNAand RNA, (2) they divide by binary fission, (3) their cell envelopesresemble those of other gram-negative bacteria, (4) they containribosomes similar to those of other bacteria, and (5) they aresusceptible to various antibiotics. Chlamydiae can be seen in the lightmicroscope, and the genome is about one-third the size of theEscherichia coli genome.

Many different strains of chlamydiae have been isolated from birds, manand other mammals, and these strains can be distinguished on the basisof host range, virulence, pathogenesis, and antigenic composition. Thereis strong homology of DNA within each species, but surprisingly littlebetween species, suggesting long-standing evolutionary separation.

C. trachomatis has a high degree of host specificity, being almostcompletely limited to man, and causes ocular and genitourinaryinfections of widely varying severity. In contrast, C. psittaci strainsare rare in man but are found in a wide range of birds and also in wild,domestic, and laboratory mammals, where they multiply in cells of manyorgans.

C. pneumoniae is a common human pathogen, originally described as theTWAR strain of C. psittaci, but subsequently recognized to be a newspecies. C. pneumoniae is antigenically, genetically, andmorphologically distinct from other Chlamydia species (C. trachomatis,C. pecorum and C. psittaci). It shows 10% or less DNA sequence homologywith either of C. trachomatis or C. psittaci and so far appears toconsist of only a single strain, TWAR.

C. pneumoniae is a common cause of community acquired pneumonia, lessfrequent only than Streptococcus pneumoniae and Mycoplasma pneumoniae(refs. 1 and 2—Throughout this application, various references arereferred to in parenthesis to more fully describe the state of the artto which this invention pertains. Full bibliographic information foreach citation is found at the end of the specification, immediatelypreceding the claims. The disclosure of these references are herebyincorporated by reference into the present disclosure). C. pneumoniaecan also cause upper respiratory tract symptoms and disease, includingbronchitis and sinusitis (refs. 1 to 4). The great majority of the adultpopulation (over 60%) has antibodies to C. pneumoniae (ref. 5),indicating past infection which was unrecognized or asymptomatic.

C. pneumoniae infection usually presents as an acute respiratory disease(i.e., cough, sore throat, hoarseness, and fever; abnormal chest soundson auscultation). For most patients, the cough persists for 2 to 6weeks, and recovery is slow. In approximately 10% of these cases, upperrespiratory tract infection is followed by bronchitis or pneumonia.Furthermore, during a C. pneumoniae epidemic, subsequent co-infectionwith pneumococcus has been noted in about half of these pneumoniapatients, particularly in the infirm and the elderly. As noted above,there is more and more evidence that C. pneumoniae infection is alsolinked to diseases other than respiratory infections.

The reservoir for the organism is presumably people. In contrast to C.psittaci infections, there is no known bird or animal reservoir.Transmission has not been clearly defined, but may result from directcontact with secretions, from formites, or from airborne spread. Thereis a long incubation period, which may last for many months. Based onanalysis of epidemics, C. pneumoniae appears to spread slowly through apopulation (case-to-case interval averaging 30 days) because infectedpersons are inefficient transmitters of the organism. Susceptibility toC. pneumoniae is universal. Reinfections occur during adulthood,following the primary infection as a child. C. pneumoniae appears to bean endemic disease throughout the world, noteworthy for superimposedintervals of increased incidence (epidemics) that persist for 2 to 3years. C. trachomatis infection does not confer cross-immunity to C.pneumoniae. Infections are easily treated with oral antibiotics,tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). Arecently developed drug, azithromycin, is highly effective as asingle-dose therapy against chlamydial infections.

In most instances, C. pneumoniae infection is mild and withoutcomplications, and up to 90% of infections are subacute or unrecognized.Among children in industrialized countries, infections have been thoughtto be rare up to the age of 5 years, although a recent study hasreported that many children in this age group show PCR evidence ofinfection despite being seronegative, and estimates a prevalence of 17to 19% in 2 to 4 years old (ref. 6). In developing countries, theseroprevalence of C. pneumoniae antibodies among young children iselevated, and there are suspicions that C. pneumoniae may be animportant cause of acute lower respiratory tract disease and mortalityfor infants and children in tropical regions of the world.

From seroprevalence studies and studies of local epidemics, the initialC. pneumoniae infection usually happens between the ages of 5 and 20years. In the USA, for example, there are estimated to be 30,000 casesof childhood pneumonia each year caused by C. pneumoniae. Infections maycluster among groups of children or young adults (e.g., school pupils ormilitary conscripts).

C. pneumoniae causes 10 to 25% of community-acquired lower respiratorytract infections (as reported from Sweden, Italy, Finland, and the USA).During an epidemic, C. pneumonia infection may account for 50 to 60% ofthe cases of pneumonia. During these periods, also, more episodes ofmixed infections with S. pneumoniae have been reported.

Reinfection during adulthood is common; the clinical presentation tendsto be milder. Based on population seroprevalence studies, there tends tobe increased exposure with age, which is particularly evident among men.Some investigators have speculated that a persistent, asymptomatic C.pneumoniae infection state is common.

In adults of middle age or older, C. pneumoniae infection may progressto chronic bronchitis and sinusitis. A study in the USA revealed thatthe incidence of pneumonia caused by C. pneumoniae in persons youngerthan 60 years is 1 case per 1,000 persons per year; but in the elderly,the disease incidence rose three-fold. C. pneumoniae infection rarelyleads to hospitalization, except in patients with an underlying illness.

Of considerable importance is the association of atherosclerosis and C.pneumoniae infection. There are several epidemiological studies showinga correlation of previous infections with C. pneumoniae and heartattacks, coronary artery and carotid artery disease (refs. 7 to 11).Moreover, the organisms has been detected in atheromas and fatty streaksof the coronary, carotid, peripheral arteries and aorta (refs. 12 to16). Viable C. pneumoniae has been recovered from the coronary andcarotid artery. (refs, 17, 18). Furthermore, it has been shown that C.pneumoniae can induce changes of atherosclerosis in a rabbit model (ref.19). Taken together, these results indicate that it is highly probablethat C. pneumoniae can cause atherosclerosis in humans, though theepidemiological importance of chlamydial atherosclerosis remains to bedemonstrated.

A number of recent studies have also indicated an association between C.pneumoniae infection and asthma. Infection has been linked to wheezing,asthmatic bronchitis, adult-onset asthma and acute exacerbation ofasthma in adults, and small-scale studies have shown that prolongedantibiotic treatment was effective at greatly reducing the severity ofthe disease in some individuals (refs. 20 to 25).

In light of these results, a protective vaccine against disease causedby C. pneumoniae infection would be of considerable importance. There isnot yet an effective vaccine for human C. pneumoniae infection.Nevertheless, studies with C. trachomatis and C. psittaci indicate thatthis is an attainable goal. For example, mice which have recovered froma lung infection with C. trachomatis are protected from infertilityinduced by a subsequent vaginal challenge (ref. 26). Similarly, sheepimmunized with inactivated C. psittaci were protected from subsequentchlamydial-induced abortions and stillbirths (ref. 27). Protection fromchlamydial infections has been associated with Th1 immune responses,particularly the induction of INFγ-producing CD4+ T cells (ref. 28). Theadoptive transfer of CD4+ cell lines or clones to nude or SCID miceconferred protection from challenge or cleared chronic disease (refs.29, 30) and in vivo depletion of CD4+ T cells exacerbated diseasepost-challenge (refs. 31, 32). However, the presence of sufficientlyhigh titres of neutralizing antibody at mucosal surfaces can also exerta protective effect (ref. 33).

The extent of antigenic variation within the species C. pneumoniae isnot well characterized. Serovars of C. trachomatis are defined on thebasis of antigenic variation in major outer membrane proteins (MOMP),but published C. pneumoniae MOMP gene sequences show no variationbetween several diverse isolates of the organism (refs. 34, 35, 36).Regions of the protein known to be conserved in other chlamydial MOMPsare conserved in C. pneumoniae (refs. 34, 35). One study has described astrain of C. pneumoniae with a MOMP of greater that usual molecularweight, but the gene for this has not been sequenced (ref. 1). Partialsequences of outer membrane protein 2 from nine diverse isolates werealso found to be invariant (ref. 17). The genes for HSP60 and HSP70 showlittle variation from other chlamydial species, as would be expected.The gene encoding a 76 kDa antigen has been cloned from a single strainof C. pneumoniae. It has no significant similarity with other knownchlamydial genes (ref. 4).

Many antigens recognized by immune sera to C. pneumoniae are conservedacross all chlamydiae, but 98kDa, 76 kDa and 54 kDa proteins may be C.pneumoniae-specific (refs. 2, 4, 37). Immunoblotting of isolates withsera from patients does show variation of blotting patterns betweenisolates, indicating that serotypes C. pneumoniae may exist (refs. 1,17). However, the results are potentially confounded by the infectionstatus of the patients, since immunoblot profiles of a patient's serachange with time post-infection. An assessment of the number andrelative frequency of any serotypes, and the defining antigens, is notyet possible.

Thus, a need remains for effective compositions for preventing andtreating Chlamydia infections.

SUMMARY OF THE INVENTION

The present invention provides a novel approach to immunizing againstChlamydial infection based on nucleic acid immunization. It hassurprisingly been found that the administration of a combination ofnucleotide sequences encoding two different chlamydial proteins providesan enhanced protection efficacy.

Accordingly, in one aspect of the present invention, there is providedan immunogenic composition for in vivo administration to a host for thegeneration in the host of a protective immune response againstChlamydial infection, comprising a first vector comprising a firstnucleotide sequence encoding a major outer membrane protein (MOMP) of astrain of Chlamydia and a first promoter sequence operatively coupled tosaid first nucleotide sequence for expression of said MOMP in the host;a second vector comprising a second nucleotide sequence encoding a 76kDa protein of a strain of Chlamydia and a second promoter sequenceoperatively coupled to said second nucleotide sequence for expression ofsaid 76 kDa protein in the host; and a pharmaceutically-acceptablecarrier therefor.

The first nucleotide sequence may encode a MOMP from any strain ofChlamydia, preferably from C. pneumoniae but also including C.trachomatis. The second nucleotide sequence encoding the MOMP protein ofC. pneumoniae may have SEQ ID No: 12, 13 or 14 or may encode a MOMPhaving a SEQ ID No: 15 or 16.

The first promoter which is employed may be a cytomegalovirus promoter,although any other convenient promoter may be employed.

The second nucleotide sequence may encode a 76 kDa protein from anystrain of Chlamydia, preferably from C. pneumoniae but also including C.trachomatis. The second nucleotide sequence encoding the 76 kDa proteinof C. pneumoniae may have SEQ ID No: 1, 2, 3 or 4. The second nucleotidesequence may encode a 76 kDa protein having a molecular size of about 35kDa and having SEQ ID No: 7 or may encode a 76 kDa protein having amolecular size of about 60 kDa and having SEQ ID No: 8 or 9.

The second promoter which is employed may be a cytomegalovirus promoter,although any other convenient promoter may be employed.

The first vector preferably comprises a plasmid vector and specificallymay be pCAMOMP. Similarly the second vector preferably comprises aplasmid vector and specifically may be pCA76 kDa. Most preferably, boththe first and second vectors are plasmid vectors and specifically thecombination of pCAMOMP and pCA76 kDa.

The two vectors are used in an immunogenic composition along with anyconvenient pharmaceutically-acceptable carrier. As noted above, the usesof the combination of two vectors produces an enhanced protectionefficacy in comparison to the individual vectors alone. Accordingly, thefirst and second vectors preferably are present in the immunogeniccomposition in amounts such that the individual protective effect ofeach vector upon administration to the composition to the host is notadversely affected by the other.

The present invention, in a further aspect thereof, provides a method ofimmunizing a host against disease caused by infection with a strain ofChlamydia, which comprises administering to the host, which may be ahuman host, an effective amount of an immunogenic composition providedherein. The immunogenic composition preferably is administeredintranasally.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the drawings, in which:

FIG. 1 shows the nucleotide sequence of C. pneumoniae 76 kDa gene (SEQID No: 1—complete sequence; SEQ ID No: 2-5′ encoding region; SEQ ID No:3-3′ encoding region including Myc and His encoding regions; SEQ ID No:4-3′ encoding region excluding Myc and His encoding regions; SEQ ID No:5—Myc encoding region; SEQ ID No: 6—His encoding region) and the deducedamino acid sequences of two open reading frames of the 76 kDa protein(SEQ ID NO: 7—upstream reading frame; SEQ ID No: 8—downstream readingframe including Myc and His regions; SEQ ID No: 9—downstream readingframe excluding Myc and His regions; SEQ ID No: 10—Myc region; SEQ IDNo: 11—His region);

FIG. 2 shows a scheme of construction of plasmid pCA76 kDa;

FIG. 3 shows the nucleotide sequence of the C. pneumoniae MOMP gene (SEQID No: 12—complete sequence; SEQ ID No: 13—encoding sequence includingMyc and His encoding regions; SEQ ID No: 14—encoding sequence excludingMyc and His encoding regions) and the deduced amino acid sequence of theMOMP protein (SEQ ID No: 15—including Myc and His regions; SEQ ID No:16—excluding Myc and His regions);

FIG. 4 shows a scheme of the construction of plasmid pCAMOMP; and

FIG. 5 illustrates the protective efficacy against C. pneumoniae lungchallenge in Balb/c mice following DNA immunization with pCAMOMP pluspCA76 kDa, in comparison to controls, wherein the individual data points(open diamonds) are shown for each animal, as well as the mean (solidsquares) and standard deviation for each group.

GENERAL DESCRIPTION OF INVENTION

As noted above, the present invention is directed to protecting a hostagainst chlamydial infection by administering to the host an immunogeniccomposition containing two vectors, preferably plasmid vectors, each ofwhich contains nucleotide sequence encoding a different protein of astrain of Chlamydia.

To illustrate the invention, a first plasmid vector was constructedcontaining the MOMP gene from C. pneumoniae and a second plasmid vectorwas constructed containing the 76 kDa protein gene from C. pneumoniae.While the invention is illustrated by the use of such plasmid vectors,other vectors containing such genes may be employed for administrationto the host for expression of the encoded proteins in the host. Suchother vectors may include live viral vectors, such as adenoviruses,alphaviruses including Semliki Forest virus and poxviruses includingavipox and canary pox viruses as well as bacterial vectors, such asShigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Bilié deCalmette-Guérin (BCG) and Streptococcus.

One of the vectors employed herein contains a nucleic acid moleculewhich codes for a Chlamydial protein known in the art as the “76 kDaprotein” (ref. 4). The latter terminology is utilized herein to refer tothe protein identified in the art. Research has determined that theencoding nucleotide sequence for this protein in fact encodes twoopening reading frames, one encoding a protein of approximately 35 kDain length (SEQ ID No: 7) and the other encoding a protein ofapproximately 60 kDa in length (SEQ ID No: 9).

It has been found that, if the complete nucleotide sequence (SEQ IDNo: 1) is incorporated into a suitable expression vector, then only the35 kDa protein is expressed. If, however, the nucleotide sequenceencoding the 60 kDa protein alone (SEQ ID No: 4) is incorporated into asuitable expression vector, then that protein also can be expressed.Both proteins have been found to be immunogenic and protective with the35 kDa protein exhibiting a stronger protective effect than the 60 kDaprotein (U.S. Patent Application No. 60/132,270 filed May 3, 1999; U.S.Patent Application No. 60/141,276 filed Jun. 30, 1999, assigned to theAssignee hereof and the disclosures of which are incorporated herein byreference).

Any convenient plasmid vector may be used for the MOMP gene and the 76kDa protein gene, such as the pcDNA3.1 expression vector (Invitrogen,San Diego, Calif., USA) containing the cytomegalovirus promoter. Schemesfor construction of the pCA76 kDa plasmid vector of 8594 bp size and ofthe pCAMOMP plasmid vector of 7.6 kb in size, which include downstreamDNA sequences coding for Myc and His tags, are shown in FIGS. 2 and 4respectively and described in detail below.

The respective plasmids are formulated into an immunogenic compositionin conjunction with a suitable pharmaceutically-acceptable carrier foradministration to a host, such as a human host. The immunogeniccomposition may be administered in any convenient manner to the host,such as intramuscularly or intranasally, although other routes ofadministration may be used, as discussed below. The data presentedherein and described in detail below demonstrates that DNA immunizationwith both the C. pneumoniae MOMP and 76 kDa protein genes elicits astrong protective immune response. The effect which is obtained isachieved without the use of adjuvant or other stimulation of immuneresponse, such as cardiotoxin, although such materials may be used, ifdesired, as discussed below. In addition, the use of immunomodulation isnot excluded from the scope of the invention. For example, it may bedesirable to coadminister DNA that expresses immunoregulator cytokines(ref. 38).

As may be seen from the data below, by utilizing both the MOMP gene andthe 76 kDa protein gene, there is obtained a protective immune responsewhich is significantly greater than that achieved using the individualgenes alone. The coadministration of the two genes does not result inany interference to the immune response of the individual genes.

There has previously been described in WO 98/02546, assigned toUniversity of Manitoba and the disclosure of which is incorporatedherein by reference, the use of the MOMP gene for DNA immunization. Theimproved results obtained herein using a combination of the MOMP geneand the 76 kDa protein gene demonstrate the use of multiple antigengenes from chlamydiae to augment the level of protective immunityachieved by DNA immunization. These results are more encouraging thanthose obtained using recombinant MOMP protein or synthetic peptides asthe immunogen.

Nucleotide sequences, e.g., DNA molecules, can easily be retrieved bypolymerase chain reaction (PCR) amplification of genomic bacterial DNAextracted by conventional methods. This involves the use of syntheticoligonucleotide primers matching upstream and downstream of the 5′ and3′ ends of the encoding domain. Suitable primers can be designedaccording to the nucleotide sequence information provided. Typically, aprimer can consist of 10 to 40, preferably 15 to 25 nucleotides. It maybe also advantageous to select primers containing C and G nucleotides ina proportion sufficient to ensure efficient hybridization; e.g., anamount of C and G nucleotides of at least 40%, preferably 50% of thetotal nucleotide amount.

It is clearly apparent to one skilled in the art that the variousembodiments of the present invention have many applications in thefields of vaccination and treatment of chlamydial infection. A furthernon-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from the MOMP gene and the 76 kDa protein gene and vectors asdisclosed herein. The vaccine elicits an immune response in a subjectwhich includes the production of anti-MOMP and anti-76 kDa proteinantibodies. Immunogenic compositions, including vaccines, containing thenucleic acid may be prepared as injectables, inphysiologically-acceptable liquid solutions or emulsions forpolynucleotide administration.

The nucleic acid may be associated with liposomes, such as lecithinliposomes or other liposomes known in the art, as a nucleic acidliposome (for example, as described in WO 93/24640) or the nucleic acidmay be associated with an adjuvant, as described in more detail below.Liposomes comprising cationic lipids interact spontaneously and rapidlywith polyanions, such as DNA and RNA, resulting in liposome/nucleic acidcomplexes that capture up to 100% of the polynucleotide. In addition,the polycationic complexes fuse with cell membranes, resulting in anintracellular delivery of polynucleotide that bypasses the degradativeenzymes of the lysosomal compartment.

Published PCT application WO 94/27435 describes compositions for geneticimmunization comprising cationic lipids and polynucleotides. Agentswhich assist in the cellular uptake of nucleic acid, such as calciumions, viral proteins and other transfection facilitating agents, mayadvantageously be used.

Polynucleotide immunogenic preparations may also be formulated asmicrocapsules, including biodegradable time-release particles. Thus,U.S. Pat. No. 5,151,264 describes a particulate carrier of aphospholipid/glycolipid/polysaccharide nature that has been termed BioVecteurs Supra Moleculaires (BVSM). The particulate carriers areintended to transport a variety of molecules having biological activityin one of the layers thereof.

U.S. Pat. No. 5,075,109 describes encapsulation of the antigenstrinitrophenylated keyhole limpet hemocyanin and staphylococcalenterotoxin B in 50:50 poly (DL-lactideco-glycolide). Other polymers forencapsulation are suggested, such as poly(glycolide),poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters andpoly(8-hydroxybutyric acid), and polyanhydrides.

Published PCT application WO 91/06282 describes a delivery vehiclecomprising a plurality of bioadhesive microspheres and antigens. Themicrospheres being of starch, gelatin, dextran, collagen or albumin.This delivery vehicle is particularly intended for the uptake of vaccineacross the nasal mucosa. The delivery vehicle may additionally containan absorption enhancer.

The vectors may be mixed with pharmaceutically acceptable excipientswhich are compatible therewith. Such excipients may include, water,saline, dextrose, glycerol, ethanol, and combinations thereof. Theimmunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof. Immunogeniccompositions and vaccines may be administered parenterally, by injectionsubcutaneously, intravenously, intradermally, intraperitoneally orintramuscularly, possibly following pretreatment of the injection sitewith a local anesthetic.

Alternatively, the immunogenic compositions formed according to thepresent invention, may be formulated and delivered in a manner to evokean immune response at mucosal surfaces. Thus, the immunogeniccomposition may be administered to mucosal surfaces by, for example, theocular, pulminary, nasal or oral (intragastric) routes. Alternatively,other modes of administration including rectal, vaginal or urinary tractas well as suppositories may be desirable. For suppositories, bindersand carriers may include, for example, polyalkylene glycols ortriglycerides. Oral formulations may include normally employedincipients, such as, for example, pharmaceutical grades of saccharine,cellulose and magnesium carbonate.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as istherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizethe MOMP and 76 kDa proteins and antibodies thereto, and if needed, toproduce a cell- mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgement of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art and may be of the order of about 1 μg to about1 mg of the vectors.

Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent administrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host. Avaccine which protects against only one pathogen is a monovalentvaccine. Vaccines which contain antigenic material of several pathogensare combined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens.

Immunogenicity may be significantly improved if the vectors areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Thus,adjuvants have been identified that enhance the immune response toantigens. Some of these adjuvants are toxic, however, and can causeundesirable side-effects, making them unsuitable for use in humans andmany animals. Indeed, only aluminum hydroxide and aluminum phosphate(collectively commonly referred to as alum) are routinely used asadjuvants in human and veterinary vaccines.

A wide range of extrinsic adjuvants and other immunomodulating materialcan provoke potent immune responses to antigens. These include saponinscomplexed to membrane protein antigens to produce immune stimulatingcomplexes (ISCOMS), pluronic polymers with mineral oil, killedmycobacteria in mineral oil, Freund's complete adjuvant, bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as Quil A derivatives and components thereof, QS 21, calciumphosphate, calcium hydroxide, zinc hydroxide, an octodecyl ester of anamino acid, ISCOPREP, DC-chol, DDBA and polyphosphazene. Advantageouscombinations of adjuvants are described in copending U.S. patentapplications Ser. No.: 08/261,194 filed Jun. 16, 1994 and Ser. No.08/483,856 filed Jun. 7, 1995, assigned to the assignee hereof and thedisclosures of which are incorporated herein by reference thereto (WO95/34308).

In particular embodiments of the present invention, the vectors may bedelivered in conjunction with a targeting molecule to target the vectorsto selected cells including cells of the immune system.

The vectors may be delivered to the host by a variety of procedures, forexample, Tang et al. (ref. 39) disclosed that introduction of goldmicroprojectiles coated with DNA encoding bovine growth hormone (BGH)into the skin of mice resulted in production of anti-BGH antibodies inthe mice, while Furth et al. (ref. 40) showed that a jet injector couldbe used to transfect skin, muscle, fat and mammary tissues of livinganimals. See also U.S. Pat. Nos. 4,245,050 and 5,015,580 and WO94/24263.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Example 1

This Example illustrates the preparation of a plasmid vector pCA76 kDacontaining the 76 kDa protein gene.

The 76 kDa protein gene was amplified from Chlamydia pneumoniae (CM1)genomic DNA by polymerase chain reaction (PCR) using a 5′ primer (5′GCTCTAGACCGCCATGACAAAAAAACAT TATGCTTGGG 3′) (SEQ ID No: 9) and 3′ primer(5′ CGGGATCCATAGAACTTGCTGCAGCGGG 3′) (SEQ ID No: 10). The 5′ primercontains a Xba I restriction site, a ribsome binding site, an initiationcodon and a sequence close to the 5′ end of the 76 kDa protein codingsequence. The 3′ primer includes the sequence encoding the C-terminalsequence of the 76 kDa protein and a Bam HI restriction site. The stopcodon was excluded and an additional nucleotide was inserted to obtainan inframe C-terminal fusion with the Histidine tag. The presence of astop codon at nucleotide 828 of the amplified sequence means that only apartial 76 kDa protein is expressed.

After amplification, the PCR fragment was using QIAquick™ PRCpurification kit (Qiagen) and then digested with Xba I and Bam HI andcloned into the pCA-Myc-His eukaryotic expression vector as described inExample 3 below (FIG. 2) with transcription under control of the humanCMV promoter.

Example 2

This Example illustrates the preparation of a plasmid vector pCAMOMPcontaining the MOMP protein gene.

The MOMP protein gene was amplified from Chlamydia pneumoniae (CM1)genomic DNA by polymerase chain reaction (PCR) using a 5′ primer (5′CCCGGATATCCCACCATGTTGCCTGTAGG GAACCCTTC 3′) (SEQ ID No: 11) and a 3′primer (5′ GGGGTACCGGAATCTGAACTGACCAGATACG 3′) (SEQ ID No: 12). The 5′primer contains a EcoRV restriction site, a ribosome binding site, aninitiation codon and a sequence encoding the N-terminal sequence of themature MOMP. The 3′ primer includes the sequence encoding the C-terminalsequence of the MOMP and a Kpn I restriction site. The DNA sequenceencoding the leader peptide was excluded, the stop codon was excludedand an additional nucleotide was inserted to obtain an in-frameC-terminal fusion with the Histdine tag.

After amplification, the PCR fragment was purified using QIAquick™ PCRpurification kit (Qiagen) and then digested with Eco RV and Kpn I andcloned into the pCA-Myc-His eukaryotic expression vector described inExample 3 (FIG. 4) with transcription under control of the human CMVpromoter.

Example 3

This Example illustrates the preparation of the eukaryotic expressionvectors pCA76 kDa and pCAMOMP.

Plasmid pcDNA3.1 (−) (Invitrogen) was restricted with Spe I and Bam HIto remove the CMV promoter and the remaining vector fragment wasisolated. The CMV promoter and intron A from plasmid VR-1012 (Vical) wasisolated on a Spe I/Bam HI fragment. The fragments were ligated togetherto produce plasmid pCA/Myc-His, as seen in FIG. 2.

The Xba I/Bam HI restricted PCR fragment containing the 76 kDa proteingene (Example 1) was ligated into the Xba I and Bam HI restrictedplasmid pCA/Myc-His to produce plasmid pCA76 kDa (FIG. 2).

The Eco RV/Kpn I restricted PCR fragment containing the MOMP gene(Example 2) was ligated into Eco RV/Kpn I restricted pCA/Myc-His toproduce plasmid pCAMOMP (FIG. 4).

The resulting plasmids, pCA76 kDa and pCAMOMP, were transferred byelectroporation into E. coli XL-1 blue (Stratagene) which was grown inLB broth containing 50 μg/ml of carbenicillin. The plasmids wereisolated by Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNApurification system. DNA concentration was determined by absorbance at260 nm and the plasmid was verified after gel electrophoresis andEthidium bromide staining and comparison to molecular weight standards.The 5′ and 3′ ends of the gene were verified by sequencing using a LiCormodel 4000 L DNA sequencer and IRD-800 labelled primers.

Example 4

This Example illustrates the immunization of mice to achieve protectionagainst an intranasal challenge by C. pneumoniae.

It has been previously demonstrated that mice are susceptible tointranasal infection with different isolates of C. pneumoniae (ref. 41).Strain AR-39 (ref. 42) was used in Balb/c mice as a challenge infectionmodel to examine the capacity of chlamydia gene products delivered asnaked DNA to elicit a protective response against a sublethal C.pneumoniae lung infection. Protective immunity is defined as anaccelerated clearance of pulmonary infection.

Groups of 7 to 9 week old male Balb/c mice (5 to 9 per group) wereimmunized intramuscularly (i.m.) and intranasally (i.n.) with plasmidspCA76 kDa and pCAMOMP containing the coding sequences of C. pneumoniae76 kDa and MOMP, respectively, prepared as described in Example 3.Saline or plasmid vectors containing non-protective inserted chlamydialgenes, namely pCAI116 and pCAI178,were given to groups of controlanimals.

The constructs pCAI116 and pCAI178 are identical to pCA76 kDa andpCAMOMP except that the nucleotide sequence encoding the partial 76 kDaprotein or MOMP is replaced with a C. pneumoniae nucleotide sequenceencoding, respectively, a possible inclusion membrane protein and anucleoside 5′-diphosphate phosphotransferase, respectively.

For i.m. immunization, alternate left and right quadriceps were injectedwith 100 μg of each DNA construct in 50 μl of PBS on three occasions at0, 3, and 6 weeks. For i.n. immunization, anaesthetized mice aspirated50 μl of PBS containing 50 μg of each DNA construct on three occasionsat 0, 3, and 6 weeks. At week 8, immunized mice were inoculated i.n.with 5×10⁵ IFU of C. pneumoniae, strain AR39, in 100 μl of SPG buffer totest their ability to limit the growth of a sublethal C. pneumoniaechallenge.

Lungs were taken from mice at day 9 post-challenge and immediatelyhomogenized in SPG buffer (7.5% sucrose, 5 mM glutamate, 12.5 mMphosphate, pH 7.5). The homogenate was stored frozen at −70° C. untilassay. Dilutions of the homogenate were assayed for the presence ofinfectious chlamydia by inoculation onto monolayers of susceptiblecells. The inoculum was centrifuged onto the cells at 3000 rpm for 1hour, then the cells were incubated for three days at 35° C. in thepresence of 1 μg/ml cycloheximide. After incubation, the monolayers werefixed with formalin and methanol, then immunoperoxidase stained for thepresence of Chlamydial inclusions using convalescent sera from rabbitsinfected with C. pneumoniae and metal-enhanced DAB as a peroxidasesubstrate.

FIG. 5 and Table 1 contain the results obtained and show that miceimmunized i.n. and i.m. with both pCA76 kDa and pCAMOMP had chlamydiallung titers less than 6700 in 6 of 6 cases, whereas the range of valuesfor control mice with saline were 15,000 to 106,100 IFU/lung in 20 outof 23 cases (mean 49,000) and 12,600 to 80,600 IFU/lung in 11 out of 12cases (mean 33,500 to 47,000) for mice immunized with the vectorscontaining non-protective genes (Table 1). The mice immunized with onlythe pCAMOMP alone showed lung titres in the range of 5800 to 18,700 in 5out of 6 cases (mean 12,600) and mice immunized with pCA76 kDa aloneshowed similar titres in the range of 6,300 to 18,200 in 5 out of 6cases (mean 7,400). The increased protection afforded by the combinationof the two constructs is surprising in light of other failures due toantigen competition. TABLE 1 BACTERIAL LOAD (INCLUSION FORMING UNITS PERLUNG) IN THE LUNGS OF BALB/C MICE IMMUNIZED WITH VARIOUS DNAIMMUNIZATION CONSTRUCTS IMMUNIZING CONSTRUCT pCAMOMP + Saline pCAI116pCAI178 pCAMOMP pCA76kDa pCA76kDa MOUSE Day 9 Day 9 Day 9 Day 9 Day 9Day 9  1 1700 47700 80600 5800 18200 6600  2 36200 12600 31900 302006300 5300  3 106100 28600 30600 9900 13400 0  4 33500 17700 6500 18700100 3300  5 70400 77300 53000 0 2400 5200  6 48700 17600 79500 110004000 2700  7 600  8 19800  9 29500 10 100000 11 15000 12 56600 13 6030014 88800 15 30400 16 69300 17 47500 18 96500 19 30200 20 84800 21 380022 65900 23 33000 MEAN 49069.57 33583.33 47016.67 12600 7400 3850 SD32120.48 24832.67 29524.32 10600.19 6981.40 2363.68

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides a novelimmunization procedure for obtaining an enhanced protective immuneresponse to Chlamydial infection by employing DNA immunization usingnucleotide sequences encoding a MOMP and a 76 kDa protein of a strain ofChlamydia. Modifications are possible within the scope of the invention.

REFERENCES

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1. An immunogenic composition for in vivo administration to a host forthe generation in the host of a protective immune response againstChlamydial infection, comprising: a first vector comprising: a firstnucleotide sequence encoding a major outer membrane protein (MOMP) of astrain of Chlamydia and a first promoter sequence operatively coupled tosaid first nucleotide sequence for expression of said MOMP in the host;a second vector comprising: a second nucleotide sequence encoding a 76kDa protein of a strain of Chlamydia and a second promoter sequenceoperatively coupled to said second nucleotide sequence for expression ofsaid 76 kDa protein in the host; and a pharmaceutically-acceptablecarrier therefor.
 2. The immunogenic composition of claim 1 wherein thefirst nucleotide sequence encodes a MOMP from Chlamydia pneumoniae. 3.The immunogenic composition of claim 1 wherein the first nucleotidesequence encodes a MOMP from Chlamydia trachomatis.
 4. The immunogeniccomposition of claim 2 wherein said first nucleotide sequence has SEQ IDNo: 12, 13 or
 14. 5. The immunogenic composition of claim 2 wherein saidfirst nucleotide sequence encodes a MOMP having SEQ ID No: 15 or
 16. 6.The immunogenic composition of claim 2 wherein the first promoter is acytomegalovirus promoter.
 7. The immunogenic composition of claim 1wherein the second nucleotide sequence encodes a 76 kDa protein fromChlamydia pneumoniae.
 8. The immunogenic composition of claim 1 whereinthe second nucleotide sequence encodes a 76 kDa protein from Chlamydiatrachomatis.
 9. The immunogenic composition of claim 7 wherein saidsecond nucleotide sequence has SEQ ID No: 1, 2, 3 or
 4. 10. Theimmunogenic composition of claim 7 wherein said second nucleotidesequence encodes a 76 kDa protein having a molecular size of about 35kDa and having SEQ ID No:
 7. 11. The immunogenic composition of claim 7wherein said second nucleotide sequence encodes a 76 kDa protein havinga molecular size of about 60 kDa and having SEQ ID No: 8 or
 9. 12. Theimmunogenic composition of claim 7 wherein said second promoter is acytomegalovirus promoter.
 13. The immunogenic composition of claim 1wherein said first vector is a plasmid vector.
 14. The immunogeniccomposition of claim 13 wherein said first plasmid vector has theidentifying characteristics of pCAMOMP as seen in FIG.
 4. 15. Theimmunogenic composition of claim 1 wherein said second vector is aplasmid vector.
 16. The immunogenic composition of claim 15 wherein saidsecond plasmid vector has the identifying characteristics of pCA76 kDaas seen in FIG.
 2. 17. The immunogenic composition of claim 1 whereinboth said first and second vectors are plasmid vectors.
 18. Theimmunogenic composition of claim 17 wherein said first plasmid vector ispCAMOMP and said second plasmid vector is pCA76 kDa.
 19. The immunogeniccomposition of claim 1 wherein said first and second vectors are presentin amounts such that the individual protective effect of each vectorupon administration of the composition to the host is not adverselyaffected by the other.
 20. The immunogenic composition of claim 1wherein said first and second vectors are present in amounts such thatan enhanced protective effect is achieved in comparison to theindividual vectors alone.
 21. A method of immunizing a host againstdisease caused by infection with a strain of Chlamydia, which comprisesadministering to said host an effective amount of an immunogeniccomposition of claim
 1. 22. The method of claim 21 wherein saidimmunogenic composition is administered intranasally.
 23. The method ofclaim 21 wherein said host is a human host.